Adeno-Associated Virus (AAV) is a leading candidate vector for human gene therapy. The long-term goal is to understand the structural determinants of capsid host interactions. This is not only of fundamental interest in virology, but is also needed to engineer recombinant viral vectors with changed cellular specificity, or enhanced immune evasion, to realize the full potential of gene therapy. With the crystallographic structure of AAV-2 in-hand and AAV-6 near completion, AAV-3 will be solved to complete structures for the predominant human serotypes. They will be used in Aim 1 as the basis of understanding the structural and sequence diversity of viral isolates in the population, and how best to mimic evolution in designing vectors resistant to immune neutralization. Aim 2 includes both functional and physical epitope mapping for AAV-2. The former will involve in vitro selection of viruses grown in the presence of monoclonal antibodies (MAb). Clusters of escape mutation sites will be mapped to the atomic structure to locate the predominant conformational epitopes. Footprints will be mapped physically through cryo-electron microscopy (EM) of complexes of AAV with MAb fragments. Aim 3 will follow our characterization of the interactions of AAV-2 with its primary cellular receptor, with characterizations of the binding of secondary receptors required for infectious entry. This will include footprinting by hydrogen-deuterium exchange analyzed by mass spectrometry, and imaging by cryo-EM. Aim 4 will continue the characterization of cellular components with which the virus interacts, now focusing on the intracellular partners bound as AAV enters through endosomes and makes its way into the nucleus. Partners will be identified using immuno-precipitation followed by mass spectrometric protein identification, opening prospects of future biophysical characterization of these interactions. Together these studies will provide a comprehensive molecular understanding of capsid-host interactions at the cellular / molecular level that will be with little precedent, and thus of fundamental interest in virology. Their elucidation is important for understanding the opportunities and constraints in engineering improved gene therapy vectors. This is a critical need if current hopes are to be realized of developing therapies for diseases with a genetic component.